Hello everyone!
I am having problem to purify protein (His tag). The protein was cloned in pET2a plasmid (commercially) expressed in E. coli (SHuffle cells) system. Few months back, I purified the protein with much more dominant band. And had good activity as well. Now I am having problem to purify the sample protein with similar purity under same condition. The nonspecific E. coli proteins are seen to be dominant which is affecting the enzyme activity. I did fresh transformation, expression, and repeat the purification but it’s not working at all. I found that the protein is not attaching in the beads (Ni-charged) after analysing the flow through in the SDS. I even bought the new SHuffle cells and tried as well. The protein expression is comparable with my previous results and is found in soluble region after cell lysis.
I wonder what had happened to the protein during the purification. Waiting for any good suggestion to optimize my work. Other His tagged proteins expressed in BL21(DE3) do not have any problem at all.
Did you used the same Ni-NTA resin you used previously? If that is the case i will suggest you to change the resin. Try to use fresh resin or you can also recharge the old one. Also check the buffers (pH) you used during purification.
Is the expression level as high as it used to be? If the level of expression is lower, the purity after a single chromatography step will likely be lower also. You could work on improving the expression and/or add a second purification step.
The purification process and the resin both are same. All the buffers are freshley prepared and recharged before purification. Regarding the expression level, there is no difference at all. The problem here is the his taged protein is not binding in the Ni-NTA resins at all.
In this case I would agree with Abhishek and try using some new Ni-NTA resin on your column. It's very possible that the Ni2+ on your column has been reduced (you can see this as the resin turns from blue to white) which means that it wont bind a his tag.
I think based on my experience that the his tag for some protein does not get exposed for Ni binding due to poor folding. You can use the flow through from your Ni chromatography and do a second purification step such as IEX to clean up the protein.
Hi. Where do you have the His tag, is it in the N-ter or the C-ter. As Kamal said about poor folding, it might happen that the His tag is cleaved off. Is there any specific reason to use SHuffle cells? If not as you said E.coli BL21 (DE3), Rosetta gami De3 and BL21-plysS are good systems as well.
If you're using a nickel column, try stripping and recharging the column, or just get a new column to use. Check pH and salt of buffers, check imidazole of buffers--too much inadvertent imidazole in binding buffers will make your protein fly right through the column.
I'd start with a freshly charged or brand new column.
Thank you everyone for your suggestion. Yes I did with new/fresh batch every thing including recharge of resin and expression. I am not getting the result. Amlan, my protein has lots of cesteine and select this ccell line (SHuffle) for proper disulfide bond formation of protein. I tried with DE3, there is good expression but the protein is inactive.
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